登革病毒4型E蛋白的表达与多克隆抗体的制备

基础医学与临床 ›› 2012, Vol. 32 ›› Issue (11): 1337-1341.

登革病毒4型E蛋白的表达与多克隆抗体的制备

李竹石¹,林华¹,杨健¹,杨会强¹,曾献武¹,赵宇¹,刘俐¹,刘莉娜¹,康庄¹,俞永新²,李玉华²

1. 成都生物制品研究所有限责任公司
2. 中国食品药品检定研究院

收稿日期:2012-06-28 修回日期:2012-09-15 出版日期:2012-11-05 发布日期:2012-10-19
通讯作者: 李玉华 E-mail:liyuhua@hotmail.com
基金资助:
国家高技术研究发展计划

Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

Received:2012-06-28 Revised:2012-09-15 Online:2012-11-05 Published:2012-10-19

摘要/Abstract

摘要： 目的在原核细胞中表达登革病毒4型E蛋白及E蛋白III区，分别以两种蛋白免疫家兔，获得可检测登革病毒的多克隆抗体。方法将登革病毒4型E蛋白与E蛋白III区编码序列克隆到pET-32a（+）质粒中，分别构建两种蛋白的表达载体，以IPTG诱导其在Rosetta细胞中的大量表达，并进行SDS-PAGE检测。分别以两种蛋白免疫家兔，制备出针对E蛋白与E蛋白III区的多克隆抗体，并对其进行Western blot鉴定。结果登革病毒4型E蛋白与E蛋白III区在Rosetta细胞中以包涵体的形式大量表达；利用所制备的多克隆抗体对登革病毒进行检测，出现了预期的条带。结论本研究制备获得的多克隆抗体可用于登革病毒的检测，为登革病毒相关研究奠定了基础。

关键词: 登革病毒4型, E蛋白, 多克隆抗体

Abstract: Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a（+） plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody could generate obvious bands as expected when reacting with the dengue virus. Conclusion The polyclonal antibody prepared is competent for the detection of dengue virus, laying a foundation for further research.

Key words: dengue virus type 4, envelope protein, polyclonal antibody

中图分类号:

Q511

李竹石林华杨健杨会强曾献武赵宇刘俐刘莉娜康庄俞永新李玉华. 登革病毒4型E蛋白的表达与多克隆抗体的制备[J]. 基础医学与临床, 2012, 32(11): 1337-1341.

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